The present invention relates generally to phosphatidylinositol 3-kinase (PI3K) enzymes, and more particularly to selective inhibitors of PI3K activity and to methods of using such materials.
Cell signaling via 3xe2x80x2-phosphorylated phosphoinositides has been implicated in a variety of cellular processes, e.g., malignant transformation, growth factor signaling, inflammation, and immunity (see Rameh et al., J. Biol Chem, 274:8347-8350 (1999) for a review). The enzyme responsible for generating these phosphorylated signaling products, phosphatidylinositol 3-kinase (PI 3-kinase; PI3K), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates phos-phatidylinositol (PI) and its phosphorylated derivatives at the 3xe2x80x2-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol 2:358-60 (1992)).
The levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primary product of PI 3-kinase activation, increase upon treatment of cells with a variety of agonists. PI 3-kinase activation, therefore, is believed to be involved in a range of cellular responses including cell growth, differentiation, and apoptosis (Parker et al., Current Biology, 5:577-99 (1995); Yao et al., Science, 267:2003-05 (1995)). Though the downstream targets of phosphorylated lipids generated following PI 3-kinase activation have not been well characterized, emerging evidence suggests that pleckstrin-homology domain- and FYVE-finger domain-containing proteins are activated when binding to various phosphatidylinositol lipids (Sternmark et al., J Cell Sci, 112:4175-83 (1999); Lemmon et al., Trends Cell Biol, 7:237-42 (1997)). In vitro, some isoforms of protein kinase C (PKC) are directly activated by PIP3, and the PKC-related protein kinase, PKB, has been shown to be activated by PI 3-kinase (Burgering et al., Nature, 376:599-602 (1995)).
Presently, the PI 3-kinase enzyme family has been divided into three classes based on their substrate specificities. Class I PI3Ks can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-biphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ks phosphorylate PI and phosphatidylinositol-4-phosphate, whereas Class III PI3Ks can only phosphorylate PI.
The initial purification and molecular cloning of PI 3-kinase revealed that it was a heterodimer consisting of p85 and p110 subunits (Otsu et al., Cell, 65:91-104 (1991); Hiles et al., Cell, 70:419-29 (1992)). Since then, four distinct Class I PI3Ks have been identified, designated PI3K xcex1, xcex2, xcex4, and xcex3, each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit. More specifically, three of the catalytic subunits, i.e., p110xcex1, p110xcex2 and p110xcex4, each interact with the same regulatory subunit, p85; whereas p110xcex3 interacts with a distinct regulatory subunit, p101. As described below, the patterns of expression of each of these PI3Ks in human cells and tissues are also distinct. Though a wealth of information has been accumulated in recent past on the cellular functions of PI 3-kinases in general, the roles played by the individual isoforms are largely unknown.
Cloning of bovine p110xcex1 has been described. This protein was identified as related to the Saccharomyces cerevisiae protein: Vps34p, a protein involved in vacuolar protein processing. The recombinant p110a product was also shown to associate with p85xcex1, to yield a PI3K activity in transfected COS-1 cells. See Hiles et al., Cell, 70, 419-29 (1992).
The cloning of a second human p110 isoform, designated p110xcex2, is described in Hu et al., Mol Cell Biol, 13:7677-88 (1993). This isoform is said to associate with p85 in cells, and to be ubiquitously expressed, as p110xcex2 mRNA has been found in numerous human and mouse tissues as well as in human umbilical vein endothelial cells, Jurkat human leukemic T cells, 293 human embryonic kidney cells, mouse 3T3 fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wide expression suggests that this isoform is broadly important in signaling pathways.
Identification of the p110xcex4 isoform of PI 3-kinase is described in Chantry et al., J Biol Chem, 272:19236-41 (1997). It was observed that the human p110xcex4 isoform is expressed in a tissue-restricted fashion. It is expressed at high levels in lymphocytes and lymphoid tissues, suggesting that the protein might play a role in PI 3-kinase-mediated signaling in the immune system. Details concerning the P1105 isoform also can be found in U.S. Pat. Nos. 5,858,753; 5,822,910; and 5,985,589. See also, Vanhaesebroeck et al., Proc Natl Acad Sci USA, 94:4330-5 (1997), and international publication WO 97/46688.
In each of the PI3xcex1, xcex2, and xcex4 subtypes, the p85 subunit acts to localize PI 3-kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins (Rameh et al., Cell, 83:821-30 (1995)). Two isoforms of p85 have been identified, p85xcex1, which is ubiquitously expressed, and p85xcex2, which is primarily found in the brain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)). Association of the p85 subunit to the PI 3-kinase p110xcex1, xcex2, or xcex4 catalytic subunits appears to be required for the catalytic activity and stability of these enzymes. In addition, the binding of Ras proteins also upregulates PI 3-kinase activity.
The cloning of p110xcex3 revealed still further complexity within the PI3K family of enzymes (Stoyanov et al., Science, 269:690-93 (1995)). The p110xcex3 isoform is closely related to p110xcex1 and p110xcex2 (45-48% identity in the catalytic domain), but as noted does not make use of p85 as a targeting subunit. Instead, p110xcex3 contains an additional domain termed a xe2x80x9cpleckstrin homology domainxe2x80x9d near its amino terminus. This domain allows interaction of p110xcex3 with the xcex2xcex3 subunits of heterotrimeric G proteins and this interaction appears to regulate its activity.
The p101 regulatory subunit for PI3Kgamma was originally cloned in swine, and the human ortholog identified subsequently (Krugmann et al., J Biol Chem, 274:17152-8 (1999)). Interaction between the N-terminal region of p101 with the N-terminal region of p110xcex3 appears to be critical for the PI3Kxcex3 activation through Gxcex2xcex3 mentioned above.
A constitutively active PI3K polypeptide is described in international publication WO 96/25488. This publication discloses preparation of a chimeric fusion protein in which a 102-residue fragment of p85 known as the inter-SH2 (iSH2) region is fused through a linker region to the N-terminus of murine p110. The p85 iSH2 domain apparently is able to activate PI3K activity in a manner comparable to intact p85 (Klippel et al., Mol Cell Biol, 14:2675-85 (1994)).
Thus, PI 3-kinases can be defined by their amino acid identity or by their activity. Additional members of this growing gene family include more distantly related lipid and protein kinases including Vps34 TOR1, and TOR2 of Saccharomyces cerevisiae (and their mammalian homologs such as FRAP and mTOR), the ataxia telangiectasia gene product (ATR) and the catalytic subunit of DNA-dependent protein kinase (DNA-PK). See generally, Hunter, Cell, 83:1-4 (1995).
PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation. A p85-associated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, which is an important costimulatory molecule for the activation of T-cells in response to antigen (Pages et al., Nature, 369:327-29 (1994); Rudd, Immunity, 4:527-34 (1996)). Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are linked to increases in the transcription of a number of genes including interleukin-2 (IL2), an important T cell growth factor (Fraser et al., Science, 251:313-16 (1991)). Mutation of CD28 such that it can no longer interact with PI 3-kinase leads to a failure to initiate IL2 production, suggesting a critical role for PI 3-kinase in T cell activation.
Specific inhibitors against individual members of a family of enzymes provide invaluable tools for deciphering functions of each enzyme. Two compounds, LY294002 and wortmannin, have been widely used as PI 3-kinase inhibitors. These compounds, however, are nonspecific PI3K inhibitors, as they do not distinguish among the four members of Class I PI 3-kinases. For example, the IC50 values of wortmannin against each of the various Class I PI 3-kinases are in the range of 1-10 nM. Similarly, the IC50 values for LY294002 against each of these PI 3-kinases is about 1 xcexcM (Fruman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited.
Based on studies using wortmannin, there is evidence that PI 3-kinase function also is required for some aspects of leukocyte signaling through G-protein coupled receptors (Thelen et al., Proc Natl Acad Sci USA, 91:4960-64 (1994)). Moreover, it has been shown that wortmannin and LY294002 block neutrophil migration and superoxide release. However, inasmuch as these compounds do not distinguish among the various isoforms of PI3K, it remains unclear which particular PI3K isoform or isoforms are involved in these phenomena. 
In view of the above considerations, it is clear that existing knowledge is lacking with respect to structural and functional features of the PI 3-kinase enzymes, including subcellular localization, activation states, substrate affinities, and the like. Moreover, the functions that these enzymes perform in both normal and diseased tissues remains to be elucidated. In particular, the function of PI3Kxcex4 in leukocytes has not previously been characterized, and knowledge concerning its function in human physiology remains limited. The coexpression in these tissues of other PI3K isoforms has heretofore confounded efforts to segregate the activities of each enzyme. Furthermore, separation of the activities of the various PI3K isozymes may not be possible without identification of inhibitors that demonstrate selective inhibition characteristics. Indeed, Applicants are not presently aware that such selective, or better, specific, inhibitors of PI3K isozymes have been demonstrated.
Thus, there exists a need in the art for further structural characterization of the PI3Kxcex4 polypeptide. There also exists a need for functional characterization of PI3Kxcex4. Furthermore, our understanding of PI3Kxcex4 requires further elaboration of the structural interactions of p110xcex4, both with its regulatory subunit and with other proteins in the cell. There also remains a need for selective or specific inhibitors of PI3K isozymes, in order that the functions of each isozyme can be better characterized. In particular, selective or specific inhibitors of PI3Kxcex4 are desirable for exploring the role of this isozyme and for development of pharmaceuticals to modulate the activity of the isozyme.
One aspect of the present invention is to provide compounds that can inhibit the biological activity of human PI3Kxcex4. Another aspect of the invention is to provide compounds that inhibit PI3Kxcex4 selectively while having relatively low inhibitory potency against the other PI3K isoforms. Another aspect of the invention is to provide methods of characterizing the function of human PI3Kxcex4. Another aspect of the invention is to provide methods of selectively modulating human PI3Kxcex4 activity, and thereby promoting medical treatment of diseases mediated by PI3Kxcex4 dysfunction. Other aspects and advantages of the invention will be readily apparent to the artisan having ordinary skill in the art.
It has now been discovered that these and other aspects can be achieved by the present invention, which, in one aspect, is a method for disrupting leukocyte function, comprising contacting leukocytes with a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3Kxcex4) activity in the leukocytes. According to the method, the leukocytes can comprise cells selected from the group consisting of neutrophils, B lymphocytes, T lymphocytes, and basophils.
For example, in cases in which the leukocytes comprise neutrophils, the method can comprise disrupting at least one neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration. Preferably, the method does not substantially disrupt bacterial phagocytosis or bacterial killing by the neutrophils. In cases wherein the leukocytes comprise B lymphocytes, the method can comprise disrupting proliferation of the B lymphocytes or antibody production by the B lymphocytes. In cases wherein the leukocytes comprise T lymphocytes, the method can comprise disrupting proliferation of the T lymphocytes. In cases wherein the leukocytes comprise basophils, the method can comprise disrupting histamine release by the basophils.
In the methods of the invention wherein a selective PI3Kxcex4 inhibitor is employed, it is preferred that the compound be at least about 10-fold selective for inhibition of PI3Kxcex4 relative to other Type I PI3K isoforms in a cell-based assay. More preferably, the compound is at least about 20-fold selective for inhibition of PI3Kxcex4 relative to other Type I PI3K isoforms in a cell-based assay. Still more preferably, the compound is at least about 50-fold selective for inhibition of PI3Kxcex4 relative to other Type I PI3K isoforms in a biochemical assay.
Preferred selective compounds useful according to the methods include compounds having the structure (I): 
wherein
A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms, and at least one ring of the system is aromatic;
X is selected from the group consisting of CHRb, CH2CHRb, and CHxe2x95x90C(Rb);
Y is selected from the group consisting of null, S, SO, SO2, NH, O, C(xe2x95x90O), OC(xe2x95x90O), C(xe2x95x90O)O, and NHC(xe2x95x90O)CH2S;
R1 and R2, independently, are selected from the group consisting of hydrogen, C1-6alkyl, aryl, heteroaryl, halo, NHC(xe2x95x90O)C1-3alkyleneN(Ra)2, NO2, ORa, OCF3, N(Ra)2, CN, OC(xe2x95x90O)Ra, C(xe2x95x90O)Ra, C(xe2x95x90O)ORa, arylORb, Het, NRaC(xe2x95x90O)C1-3alkyleneC(xe2x95x90O)ORa, arylOC1-3alkylene-N(Ra)2, arylOC(xe2x95x90O)Ra, C1-4alkyleneC(xe2x95x90O)ORa, OC1-4alkyl-eneC(xe2x95x90O)ORa, C1-4alkyleneOC1-4alkyleneC(xe2x95x90O)ORa, C(xe2x95x90O)xe2x80x94NRaSO2Ra, C1-4alkyleneN(Ra)2, C2-6alkenyleneN(Ra)2, C(xe2x95x90O)NRaC1-4alkyleneORa, C(xe2x95x90O)NRaC1-4alkyleneHet, OC2-4-alkyleneN(Ra)2, OC1-4alkyleneCH(ORb)CH2N(Ra)2, OC1-4alkyleneHet, OC2-4alkyleneORa, OC2-4alkyleneNRaC(xe2x95x90O)ORa, NRaC1-4alkyleneN(Ra)2, NRaC(xe2x95x90O)Ra, NRaC(xe2x95x90O)N(Ra)2, N(SO2C1-4alkyl)2, NRa(SO2C1-4alkyl), SO2N(Ra)2, OSO2CF3, C1-3alkylenearyl, C1-4alkyleneHet, C1-6alkyleneORb, C1-3alkyleneN(Ra)2, C(xe2x95x90O)N(Ra)2, NHC(xe2x95x90O)C1-C3alkylenearyl, C3-8cycloalkyl, C3-8heterocycloalkyl, arylOC1-3alkyleneN(Ra)2, arylOC(xe2x95x90O)Rb, NHC(xe2x95x90O)C1-3alkyleneC3-8heterocycloalkyl, NHC(xe2x95x90O)C1-3alkyleneHet, OC1-4alkyleneOC1-4alkyleneC(xe2x95x90O)ORb, C(xe2x95x90O)C1-4alkyleneHet, and NHC(xe2x95x90O)haloC1-6alkyl;
or R1 and R2 are taken together to form a 3- or 4-membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;
R3 is selected from the group consisting of optionally substituted hydrogen, C1-6alkyl, C3-8cyclo-alkyl, C3-8heterocycloalkyl, C1-4alkylenecycloalkyl, C2-6alkenyl, C1-3alkylenearyl, arylC1-3alkyl, C(xe2x95x90O)Ra, aryl, heteroaryl, C(xe2x95x90O)ORa, C(xe2x95x90O)N(Ra)2, C(xe2x95x90S)N(Ra)2, SO2Ra, SO2N(Ra)2, S(xe2x95x90O)Ra, S(xe2x95x90O)N(Ra)2, C(xe2x95x90O)NRaC1-4alkyleneORa, C(xe2x95x90O)NRaC1-4alkyleneHet, C(xe2x95x90O)C1-4alkylenearyl, C(xe2x95x90O)C1-4alkyleneheteroaryl, C1-4alkylenearyl optionally substituted with one or more of halo, SO2N(Ra)2, N(Ra)2, C(xe2x95x90O)ORa, NRaSO2CF3, CN, NO2, C(xe2x95x90O)Ra, ORa, C1-4alkyleneN(Ra)2, and OC1-4alkyleneN(Ra)2, C1-4alkyleneheteroaryl, C1-4alkyleneHet, C1-4alkyleneC(xe2x95x90O)xe2x80x94C1-4alkylenearyl, C1-4alkyleneC(xe2x95x90O)C1-4alkyleneheteroaryl, C1-4alkyleneC(xe2x95x90O)Het, C1-4alkyleneC(xe2x95x90O)N(Ra)2, C1-4alkyleneoRa, C1-4alkyleneNRaC(xe2x95x90O)Ra, C1-4alkyleneOC1-4alkyleneORa, C1-4alkyleneN(Ra)2, C1-4alkyleneC(xe2x95x90O)xe2x80x94ORa, and C1-4alkyleneOC1-4alkyleneC(xe2x95x90O)ORa;
Ra is selected from the group consisting of hydrogen, C1-6alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C1-3alkyleneN(Ra)2, aryl, arylC1-3alkyl, C1-3alkylenearyl, heteroaryl, heteroarylC1-3alkyl, and C1-3alkyleneheteroaryl;
or two Ra groups are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;
Rb is selected from the group consisting of hydrogen, C1-6alkyl, aryl, heteroaryl, arylC1-3alkyl, heteroarylC1-3alkyl, C1-3alkylenearyl, and C1-3alkyleneheteroaryl;
Het is a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with C1-4alkyl or C(xe2x95x90O)ORa;
and pharmaceutically acceptable salts and solvates (e.g., hydrates) thereof,
wherein the compound has at least about a 10-fold selective inhibition for PI3Kxcex4 relative other Type-I PI3K isoforms in a cell-based assay.
In another embodiment, the invention is a method for treating a medical condition mediated by neutrophils, comprising administering to an animal in need thereof an effective amount of a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3Kxcex4) activity in the neutrophils. Exemplary medical conditions that can be treated according to the method include those conditions characterized by an undesirable neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration. Preferably, according to the method, phagocytic activity or bacterial killing by the neutrophils is substantially uninhibited.
In another embodiment, the invention is a method for disrupting a function of osteoclasts comprising contacting osteoclasts with a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3Kxcex4) activity in the osteoclasts. According to the method, the compound can comprise a moiety that preferentially binds to bone.
In another embodiment, the invention is a method of ameliorating a bone-resorption disorder in an animal in need thereof comprising administering to the animal an effective amount of a compound that inhibits phosphatidylinositol 3-kinase delta (PI3Kxcex4) activity in osteoclasts of the animal. A preferred bone-resorption disorder amenable to treatment according to the method is osteoporosis.
In another embodiment, the invention is a method for inhibiting the growth or proliferation of cancer cells of hematopoietic origin, comprising contacting the cancer cells with a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3Kxcex4) activity in the cancer cells. The method can be advantageous in inhibiting the growth or proliferation of cancers selected from the group consisting of lymphomas, multiple myelomas, and leukemias.
In another embodiment, the invention is a method of inhibiting kinase activity of a phosphatidylinositol 3-kinase delta (PI3Kxcex4) polypeptide, comprising contacting the PI3Kxcex4 polypeptide with a compound having the generic structure (I).
Preferred compounds useful according to the method include compounds selected from the group consisting of: 
wherein
Y is selected from the group consisting of null, S, and NH;
R4 is selected from the group consisting of H, halogen, NO2, OH, OCH3, CH3, and CF3;
R5 is selected from the group consisting of H, OCH3, and halo;
or R4 and R5 together with C-6 and C-7 of the quinazoline ring system define a 5- or 6-membered aromatic ring optionally containing one or more O, N, or S atoms;
R6 is selected from the group consisting of C1-C6alkyl, phenyl, halophenyl, alkoxyphenyl, alkyl-phenyl, biphenyl, benzyl, pyridinyl, 4-methylpiper-azinyl, C(xe2x95x90O)OC2H5, and morpholinyl;
Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl), and 
q is 1 or 2,
provided that at least one of R4 and R5 is other than H when R6 is phenyl or 2-chlorophenyl.
More preferably, the compound is selected from the group consisting of: 
wherein
Y is selected from the group consisting of null, S, and NH;
R7 is selected from the group consisting of H, halo, OH, OCH3, CH3, and CF3;
R8 is selected from the group consisting of is H, OCH3, and halogen;
or R7 and R8 together with C-6 and C-7 of the quinazoline ring system define a 5- or 6-membered aromatic ring optionally containing one or more O, N, or S atoms;
R9 is selected from the group consisting of C1-C6alkyl, phenyl, halophenyl, alkylphenyl, biphenyl, benzyl, pyridinyl, 4-methylpiperazinyl, C(xe2x95x90O)OC2H5, and morpholinyl;
Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl); and
q is 1 or 2,
provided that at least one of R7 and R8 is different from 6-halo or 6,7-dimethoxy groups, and that R9 is different from 4-chlorophenyl.
In another embodiment, the invention is a method for disrupting leukocyte function, comprising contacting leukocytes with a compound having a general structure (I).
In another embodiment, the invention is a class of compounds that have been observed to inhibit PI3Kxcex4 activity in biochemical and cell-based assays, and are expected to exhibit therapeutic benefit in medical conditions in which PI3Kxcex4 activity is excessive or undesirable. Thus, the invention provides a class of compounds having the structure (II).
Preferably, the compounds have a general structure (IV). 
wherein
Y is selected from the group consisting of null, S, and NH;
R10 is selected from the group consisting of H, halo, OH, OCH3, CH3, and CF3;
R11 is selected from the group consisting of H, OCH3, and halo;
or R10 and R11 together with C-6 and C-7 of the quinazoline ring system define a 5- or 6-membered aromatic ring optionally containing one or more O, N, or S atoms;
R12 is selected from the group consisting of C1-C6alkyl, phenyl, halophenyl, alkylphenyl, biphenyl, benzyl, pyridinyl, 4-methylpiperazinyl, C(xe2x95x90O)C2H5, and morpholinyl;
Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl), and
q is 1 or 2,
provided that:
(a) at least one of R10 and R11 is different from 6-halo or 6,7-dimethoxy groups;
(b) R12 is different from 4-chlorophenyl; and
(c) at least one of R10 and R11 is different from H when R12 is phenyl or 2-chlorophenyl and X is S.
These and other features and advantages of the present invention will be appreciated from the detailed description and examples that are set forth herein. The detailed description and examples are provided to enhance the understanding of the invention, but are not intended to limit the scope of the invention.